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Beneficial Uses of Viruses in Biotechnology. Gene therapy Vaccines and vaccine carrier / delivery vehicles Antibacterial agents Basic knowledge of cell metabolic processes Vectors for mammalian, plant and insect cell protein expression systems

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beneficial uses of viruses in biotechnology
Beneficial Uses of Viruses in Biotechnology
  • Gene therapy
  • Vaccines and vaccine carrier / delivery vehicles
  • Antibacterial agents
  • Basic knowledge of cell metabolic processes
  • Vectors for mammalian, plant and insect cell protein expression systems
  • Peptide display – industrial /pharmaceutical / medical reagent development
gene therapy viruses as gene delivery vehicles
Gene therapy: Viruses as gene delivery vehicles
  • Necessary viral properties
    • Use a virus that persists in humans, and shows moderate level of long-term gene expression
    • Need to clone in foreign DNA (where, how much) – develop cloning sites, need large cloning capacity
    • Virus must target specific cell-type – cell-type-specific receptor mediated attachment and uptake – can genetically engineer for receptor
slide3

Gene therapy: Viruses as gene delivery vehicles

  • Manufacture
  • propagate virus in specific mammalian cell line – expensive
  • Biosafety - mutate to produce attenuated virus (low virulence) or crippled virus (reduced pathogenicity) - but generally low titre
  • To circumvent low titre – Helper virus or transgenic cell line provides packaging / replication functions in trans for non-replicating delivery virus
  • viral packaging - protects and stabilises DNA from degradation
slide4

Gene therapy: Viruses as gene delivery vehicles

  • Host response
    • Do not want a strong immune response to viral vector (& its associated payload) – leads to rapid clearance of delivery virus
    • Thus, use viruses with
    • rare serotypes
    • low seroprevalence
    • low-level replication or non-replicating virus
    • E.g. lentiviruses
viruses direct use in vaccines
Viruses: Direct use in vaccines
  • Against autologous virus
    • attenuated or inactivated virus
    • viral subunits - usually structural proteins, genetic vaccines
    • cross-reactivity - study relatedness of virus strains
  • Against heterologous virus
    • Viral structural proteins or “virus-like particles” (VLPs) can be made to carry heterologous pathogen epitopes
    • Must produce proteins, VLPs in quantity to high purity
    • e.g. rabies virus glycoprotein, HPV
viruses as dna vaccine delivery vehicles
Viruses as DNA vaccine delivery vehicles
  • Preferred Immunological Properties
    • Want weak immune response (IR) to viral delivery vehicle
    • Weak / moderate IR to delivery vehicle results in enhanced response to DNA payload
    • Strong IR to delivery vehicle may provoke toxic over-response, clear the vaccine too rapidly for a response to develop to the payload, or can swamp response to payload
    • Therefore:
    • Use viruses having rare serotypes (low seroprevalence)
    • low virulence or non-pathogenic viruses
viruses as dna vaccine delivery vehicles7
Viruses as DNA vaccine delivery vehicles
  • Develop a viral delivery vehicle
    • study gene function, engineer suitable cloning sites
    • amount of DNA vaccine that can be cloned and packaged is limited by capsid size / viral packaging mechanism
  • Cell targeting – DNA delivery
    • viral engulfment by antigen presenting cells
    • cell-specific receptor-mediated uptake
  • Manufacture
    • Prefer a virus that replicates to high titre
    • Prefer a virus with a long survival half life outside host cell
    • Viral packaging of DNA vaccine protects and stabilises DNA from degradation
    • Need suitable production-host cells – if using attenuated virus (eg vaccinia vectors) - may need to provide some packaging / replication functions in trans
slide8

Plasmid DNA

makes encoded HIV protein in cells of the body

Vector Vaccines for HIV-1

Virus-like particle with outer surface display of epitopes

Epitope Display Vectors

Live AttenuatedViral

Vectors

Adenovirus

Modified Vaccinia (MVA)

Replicon Vaccines:

Virally encapsidated plasmid vaccine

DNA from HIV is Cloned

into Various Vectors

slide9

Plasmid DNA

makes encoded HIV protein in cells of the body

Vector Vaccines for HIV-1

DNA from HIV is Cloned

into Various Vectors

viral g enetic elements used to construct eukaryotic expression plasmid vectors
Viral genetic elements used to construct Eukaryotic expression plasmid vectors
  • Viruses are highly efficient replicators & viral gene expression is adapted to eukaryotic systems
    • very strong promoters (CMV immediate / early promoter)
    • small introns (CMV intron)
    • regulatory elements often constitutive - require only host factor binding (porcine circovirus (PCV) capsid promoter / enhancer)
  • Therefore mine regulatory elements from viruses
    • Promoters, enhancers, polyadenylation signals, introns, replication origins, IRES elements.
slide11

Vector Vaccines for HIV-1

Virus-like particle with outer surface display of epitopes

Epitope Display Vectors

DNA from HIV is Cloned

into Various Vectors

slide12

Use of insect Baculovirus :

Autographa californica nuclear polyhedrosis virus (AcNPV):

Foreign gene (HIV-1 gag) inserted under Baculovirus strong late promoter, polh -transient production of HIV Virus-Like Particles in cultured insect cells

A. Meyers, E.P. Rybicki.

slide13

Tobacco mosaic virus

Viruses for Peptide display: M13 Phage or plant virus (TMV) Coat Protein Fusions

  • Need :
  • non-enveloped virus
  • many repeat capsid subunits
  • ordered capsid array - amplified display
  • external loops or termini available for peptide addition via gene fusion

Mass peptide display on outer surface of TMV particle

N

C

60S loop

Assembly of mixed TMV capsids carrying epitope variants = useful vaccine vs highly variable pathogen

TMV VIRION

slide14

Vector Vaccines for HIV-1

Live AttenuatedViral

Vectors

Adenovirus

Modified Vaccinia (MVA)

DNA from HIV is Cloned

into Various Vectors

slide15

Live Attenuated Viral Vectors at UCT

Modified Vaccinia Ankara (MVA)

  • HIV-1 vaccine development at UCT
  • Recombinant MVA (rMVA) expressing HIV-1C gag and env genes
  • Used in a Prime-Boost immunisation regimen
    • prime immune response with plasmid vaccine expressing gag and env
    • boost to broaden / increase response with rMVA expressing gag and env

DNA prime

rMVA boost

slide16

Vector Vaccines for HIV-1

Replicon Vaccines:

Virally encapsidated plasmid vaccine

DNA from HIV is Cloned

into Various Vectors

replicon vaccines virally encapsidated plasmid vaccine
Replicon Vaccines: Virally encapsidated plasmid vaccine
  • Adenovirus 5, Adeno-associated virus
  • Bacteriophage vectors e.g. Lambda or M13
    • clone foreign DNA into Lambda genome
    • Large cloning capacity
    • passive uptake by immune cells and complement mediated uptake
    • Non-pathogenic for humans - safe
    • Highly stable vehicle - can dehydrate
    • Cheap to make – high titre production in E. coli
principle

Macrophage

Dendritic cell

Cell transcribes DNA.

Vaccine protein is expressed on cell surface

Principle:

Mammalian expression control elements

l DNA

l DNA

Antigen gene

Phage broken down.

Vaccine-encoding DNA released

Vaccine expression cassette cloned into bacteriophage l DNA

Immune response

Grow l phage in E. coli & purify

Antigen – presenting cells engulf l particles

Inoculate - injection / oral

bacteriophage viral antibacterial agents
Bacteriophage: viral antibacterial agents
  • Advantages:
    • Useful where multiple antibiotic resistance has developed
    • host specific - won't kill off commensal bacteria
    • Rapid action – exponential replication
    • self-limiting infection once pathogenic bacteria are killed
    • cheap - single dose - self propagates
  • Disadvantage - strain specific
    • need to generate, keep and archive large bank of phage serotypes
    • need accurate diagnosis
    • must give cocktail of phage types to prevent bacterial escape
  • Also use for detecting pathogenic bacteria - phage infects bacterial lawn - assay plaques by antibody or by phage-encoded marker gene expression

Multi drug resistant Pseudomonas